The neocortex is comprised of a dense population of excitatory neurons that are balanced by a diverse and more sparsely distributed array of inhibitory neurons. As such, cortical inhibitory neurons provide a gain control that works under a myriad of conditions. In primary visual cortex (V1), for example, inhibition is essential for severl basic functional characteristics of individual neurons, including preferences for stimulus contrast, size, and orientation. While inhibitory neurons serve to control excitation it is not knon whether inhibitory and excitatory cell-types are mediated through the same set of cortical circuits, nor whether the functional selectivity of these circuits differ. Determining the origins f the neural circuits to these two major cell-types will provide tremendous insight into the basic mechanisms of cortical processing. However, in order to do so it is necessary to develop a technique for cell-type specific neuroanatomical tracing. The proposal here is to develop such a technique by taking advantage of the neurotropism of recombinant viral vectors (adeno associated virus and lentivirus) and selectivity of cell-type specific promoters (GAD-67 and -CamKII) to deliver and allow for expression of two key genes (TVA and RabG). These genes will thereby be selectively expressed in either inhibitory or excitatory neocortical cells, but not both. The TVA and RabG proteins expressed in these specific cell types will allow for targeted viral infection and trans-complementation of a novel genetically modified and pseudo-typed rabies virus (EnvA-RabG) that acts as a monosynaptic retrograde tracer (Wickersham, Lyon et al., 2007, Neuron). This highly innovative combination of cell-type specific expression of these genes and the targeted selectivity of the EnvA-RabG rabies virus will enable independent tracing of the connections of local clusters of inhibitory or excitatory cells. Moreover, once developed it will be used to trace the long-range connectivity of inhibitory and excitatory neurons in cat V1 with respect to the orientation map; maps that will be derived through intrinsic signal optical imaging. In this way, the functional preference of inputs to inhibitory neurons in neocortex can be determined for the first time and compared to the preference of inputs to excitatory cell populations. Finally, while the goal of this proposal is to ultimately implement th new technique to understand intrinsic circuits in cat V1, this technique will be available to other for use in any mammalian species and any region of neocortex. PUBLIC HEALTH RELEVANCE: The method proposed to be developed here will allow for selective neuroanatomical tracing of inputs to the two major classes of neurons in the mammalian brain, inhibitory and excitatory, rather than indiscriminant tracing of both types together, as current techniques do. This method involves the development and combination of a number of highly innovative tools including recombinant viruses, cell- type specific promoters, a genetically modified and pseudo-typed rabies virus, and intrinsic signal optical imaging. Once developed the technique can be used in any region of neocortex to determine, for the first time, how neural circuits of inhibitory and excitatory neurons compare and then relate these circuits to differences in cell type function.